B. ] ENNESLAND, T. NAKAMOTO, AND B. STERN 613 



were rather didcrent. This proinpted us to see whether we might 

 not be able to (onfirni some of the other fiiuHngs reported by War- 

 burg and his collaborators. Because ol its theoretical importance, 

 the report that CX)^, is required tor the photoevolution of Oo seemed 

 particularly interesting. We therefore made efforts to confirm the 

 statement that the Hill reaction is stimulated by CO^. 



For a ninnber of [practical reasons, we chose to search for a CO2 

 effect with a system in which spinach chloroplasts photoreduce ferri- 

 cyanide in the presence of a catalytic amount of trichlorophenol in- 

 dophenol. The data shown in Table 5 were obtained by Babette 

 Stern. They are presented as evidence that COo (or bicarbonate) 

 stimulates the rate of photoreduction in the particular system chosen. 

 The incubations were carried out in double-arm Warburg vessels, 

 and pressure changes were followed manometrically. The gas phase 

 was air, which was either freed of CO2 by the use of base in the 

 center-well or enriched with COo to a sufficient extent to give about 

 2 yumoles of bicarbonate in the liquid medium (compared with 100 

 /xmoles of phosphate) . Each reaction mixture contained 0.2 mg 

 chlorophyll in the form of grana prepared by treating spinach chloro- 

 plasts with water. The grana were suspended in a phosphate-chloride 

 medium and added to one side-arm. The other side-arm contained 

 trichloroacetic acid, which was tipped in to stop the reaction at the 

 end of the incubation period. This provided a check on the carbonate 

 content of the medium. The preincubation period listed in the left 



TABLE 5 

 Effect of Carbonate on Ferricyanide Reduction 



Reaction mixtures contained 100 /umoles of phosphate buffer of pH 7.0, 0.1% KCl, 

 0.1 umole trichlorophenol indophenol, 20 ^moles ferricyanide, and spinach grana 

 containing 0.2 mg chlorophyll, in a volume of 3.0 ml. Grana added from side-arm 

 after preincubation in dark. Temp. 15°C; white light. 



